Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Nov 14;20(22):5719.
doi: 10.3390/ijms20225719.

Hsp40 Protein DNAJB6 Interacts with Viral NS3 and Inhibits the Replication of the Japanese Encephalitis Virus

Yu-Qin Cao  1   2 , Lei Yuan  3 , Qin Zhao  1   2   4 , Jian-Lin Yuan  1   2 , Chang Miao  1   2 , Yung-Fu Chang  5 , Xin-Tian Wen  1   2 , Rui Wu  1   2 , Xiao-Bo Huang  1   2 , Yi-Ping Wen  1   2 , Qi-Gui Yan  1   2 , Yong Huang  1   2 , Xin-Feng Han  1   2 , Xiao-Ping Ma  1   2 , San-Jie Cao  1   2   4
Affiliations

Hsp40 Protein DNAJB6 Interacts with Viral NS3 and Inhibits the Replication of the Japanese Encephalitis Virus

Yu-Qin Cao et al. Int J Mol Sci. .

Abstract

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus prevalent in east and southeast Asia, the Western Pacific, and northern Australia. Since viruses are obligatory intracellular pathogens, the dynamic processes of viral entry, replication, and assembly are dependent on numerous host-pathogen interactions. Efforts to identify JEV-interacting host factors are ongoing because their identification and characterization remain incomplete. Three enzymatic activities of flavivirus non-structural protein 3 (NS3), including serine protease, RNA helicase, and triphosphatase, play major roles in the flaviviruses lifecycle. To identify cellular factors that interact with NS3, we screened a human brain cDNA library using a yeast two-hybrid assay, and identified eight proteins that putatively interact with NS3: COPS5, FBLN5, PPP2CB, CRBN, DNAJB6, UBE2N, ZNF350, and GPR137B. We demonstrated that the DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6) colocalizes and interacts with NS3, and has a negative regulatory function in JEV replication. We also show that loss of DNAJB6 function results in significantly increased viral replication, but does not affect viral binding or internalization. Moreover, the time-course of DNAJB6 disruption during JEV infection varies in a viral load-dependent manner, suggesting that JEV targets this host chaperone protein for viral benefit. Deciphering the modes of NS3-interacting host proteins functions in virion production will shed light on JEV pathogenic mechanisms and may also reveal new avenues for antiviral therapeutics.

Keywords: Japanese encephalitis virus; flavivirus; non-structural protein 3 (NS3); replication; the DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6); virus–host interactions.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
A yeast two-hybrid screen of a human brain cDNA library reveals NS3-interacting host factors. The yeast strain Y2HGold was co-transformed with the prey plasmid AD-host factors and the bait plasmid BD or BD-NS3. Co-transformation with BD/AD, BD-Lamin/AD-T, and BD-p53/AD-T were used as blank, negative, and positive controls, respectively. The screen yielded eight host factors that potentially interact with NS3.
Figure 2
Figure 2
NS3-DNAJB6 interaction verified by co-immunoprecipitation and confocal immunofluorescence microscopy. (A) HEK293T cells were co-transfected with a GFP-tagged NS3 expression plasmid with or without HA-DNAJB6 for 48 h. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates and whole cell lysates were analyzed by Western blot using anti-DNAJB6 or anti-NS3. (B) DNAJB6 colocalizes with JEV NS3 in HEK293 cells. HEK293 cells transfected with plasmids expressing HA-DNAJB6 for 24 h were infected with JEV for another 36 h. These were subjected to immunofluorescence assay using anti-DNAJB6 and anti-NS3 antibodies, followed by Alexa 488 anti-rabbit and Alexa 555 anti-mouse antibodies. Cells were imaged on a confocal microscope. Magnification, ×600. Bars, 20μm. Z-stacks were acquired by sequential scanning. Abbreviations: IP: immunoprecipitation; IB, immunoblotting.
Figure 3
Figure 3
Overexpression of DNAJB6 inhibits JEV infection. (A) Western blot analysis of HEK293 cells overexpressing DNAJB6. The relative DNAJB6 levels from the cells before they used for subsequent experiments were all determined using Western blot analysis to ensure DNAJB6 was overexpressed. The mock (untransfected) and control (transfected with empty vector) lanes illustrate the level of endogenous expression of DNAJB6. (B–D) Infection assays of HEK293 cells overexpressing DNAJB6 then infected with JEV at MOI of 1.0 for 24 h and/or 48 h. (B) JEV infection measured by NS3 protein (green) immunofluorescence. Scale bar, 100 µm. Quantitation of the JEV NS3 signal integrated density was normalized to the control cells (Mean ± SD, n = 3, Student’s t test; *** p < 0.001). (C) Viral mRNA levels measured by qRT-PCR (Mean ± SD, n = 3, Student’s t test; * p < 0.05, ns, not significant). (D) JEV titers measured by plaque assay (Mean ± SD, n = 3, one-way ANOVA; ** p < 0.01). (E) SK-N-SH cells overexpressing DNAJB6 then infected with JEV at MOI of 1.0 for 48 h. JEV titers were determined by plaque assay (Mean ± SD, n = 3, Student’s t test; ** p < 0.01).
Figure 4
Figure 4
Generation and validation of DNAJB6 knockout cells. (A) Illustration of the disrupted alleles of DNAJB6 in HEK293 cells using CRISPR/Cas9. (B) DNAJB6 knockout in cell clones was verified by Western blot, wild type (WT) HEK293 cells are the control. (C) Cell viability assays based on quantitation of ATP. ΔDNAJB6 and parental cells were seeded at 5 × 103 or 1 × 104 cells per well in 96-well plates in DMEM/10% FBS. Luminescence was recorded 10 min after reagent addition. (Mean ± SD, n = 3, Student’s t test; ns, not significant).
Figure 5
Figure 5
Effect of the loss of DNAJB6 on propagation of JEV. (A–C) Knocking out host factor DNAJB6 results in increased JEV propagation. ΔDNAJB6 and parental cells were infected with JEV at MOI of 1.0. At 24 and 48 hpi, JEV infection measured by (A) plaque assay for viral titers, (B) immunofluorescence for viral NS3 protein (red) expression, scale bar = 100 µm, and (C) qRT-PCR for viral mRNA levels. Quantitation of the NS3 signal integrated density normalized to the control is provided. (D–F) Expression of human DNAJB6 in ΔDNAJB6 cells resulted in partially restored anti-JEV activity. ΔDNAJB6 and parental cells were transfected with the DNAJB6 expressing plasmid or empty vector, followed by infection with JEV at MOI of 1.0. At 24 and 48 hpi JEV infection measured by (D) qRT-PCR for viral mRNA levels, (E) plaque assay for viral titers, and (F) Western blot for NS3 expression, GAPDH was used as an internal control. (Mean ± SD, n = 3, Student’s t test; * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant).
Figure 6
Figure 6
DNAJB6 inhibits JEV replication but does not affect viral entry. (A) Detection of the expression level of replicon NS3 in ΔDNAJB6 cells and control cells. 2 μg of in vitro-transcribed replicon RNA was transfected into 106 ΔDNAJB6 and parental cells, lysates were collected at the indicated times post transfection and the NS3 expression was analyzed by Western blot. GAPDH was used as an internal control. (B) Luciferase activity of JEV-Rluc-Rep in ΔDNAJB6 and parental cells. Cells were treated as described above; luciferase assays were performed at the indicated time points post transfection (Mean ± SD, n = 3, two-way ANOVA; *** p < 0.001). (C) Role of DNAJB6 in JEV binding and internalization into cells. Virus binding to and internalization into cells were measured by qRT-PCR of JEV RNA (Mean ± SD, n = 3, Student’s t test, ns, not significant).
Figure 7
Figure 7
JEV infection downregulates the expression of DNAJB6. (A) DNAJB6 mRNA levels in mock and JEV infected (MOI of 1.0) HEK293 cells. The total cellular RNA was extracted and the levels of DNAJB6 were determined by RT-qPCR and normalized to β-actin at each time point. (B-C) DNAJB6 protein levels in mock and JEV infected HEK293 cells. (B) Western blot was performed to examine the expression of the cellular DNAJB6 protein, GAPDH was used as a loading control. (C) Quantitation of DNAJB6 protein normalized to the GAPDH. (Mean ± SD, n = 3, two-way ANOVA; *** p < 0.001).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Daep C.A., Munoz-Jordan J.L., Eugenin E.A. Flaviviruses, an expanding threat in public health: Focus on dengue, West Nile, and Japanese encephalitis virus. J. Neurovirol. 2014;20:539–560. doi: 10.1007/s13365-014-0285-z. - DOI - PMC - PubMed
    1. Geiss B.J., Stahla H., Hannah A.M., Gari A.M., Keenan S.M. Focus on flaviviruses: Current and future drug targets. Future Med. Chem. 2009;1:327–344. doi: 10.4155/fmc.09.27. - DOI - PMC - PubMed
    1. Solomon T., Ni H., Beasley D.W., Ekkelenkamp M., Cardosa M.J., Barrett A.D. Origin and evolution of Japanese encephalitis virus in southeast Asia. J. Virol. 2003;77:3091–3098. doi: 10.1128/JVI.77.5.3091-3098.2003. - DOI - PMC - PubMed
    1. Huang S.H., Lien J.C., Chen C.J., Liu Y.C., Wang C.Y., Ping C.F., Lin Y.F., Huang A.C., Lin C.W. Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload. Int. J. Mol. Sci. 2016;17:1386. doi: 10.3390/ijms17091386. - DOI - PMC - PubMed
    1. Misra U.K., Kalita J. Overview: Japanese encephalitis. Prog. Neurobiol. 2010;91:108–120. doi: 10.1016/j.pneurobio.2010.01.008. - DOI - PubMed

MeSH terms

LinkOut - more resources

-